A critical roadblock in the development of an HIV-1 vaccine is our current inability to deliver HIV-1 antigens efficiently to the immune system and to prime predictable, high frequency immune responses in humans. Recombinant Ad5 vector-based vaccines have elicited potent immune responses in preclinical studies. However, the high frequency of anti-Ad5 immunity in the developing world will likely substantially limit the immunogenicity and clinical utility of rAd5 vaccines. We therefore propose the development of novel, replication-incompetent rAd vector-based vaccines for HIV-1. The immunogenicity of several novel rAd vaccines will be assessed in rhesus monkeys with anti-Ad5 immunity in Project 1 of this IPCAVD grant, and the optimal vector combination will be advanced into the clinical studies described in this project. We predict that the most immunogenic rAd vaccine regimen will involve two heterologous rAd vectors that are both rare in human populations, engineered for optimal immunogenicity, derived from different Ad subfamilies, and distinct from Ad5. We assume that this will be the rAd35k5-rAd49 prime-boost regimen. Clinical-grade vaccine vectors will be manufactured and regulatory approvals will be obtained through the Vector Core Facility (Core B). In this project, we propose to investigate the hypothesis that the optimal heterologous rAd primeboost regimen will prove safe and highly immunogenic in individuals both in the United States and in the developing world. We further hypothesize that the immunogenicity of these vaccines will not be substantially suppressed by anti-vector immunity present in human populations. To investigate these hypotheses, we propose the following three Specific Aims: 1. To perform a phase I dose-escalation study to assess the safety and immunogenicity of the rAd35k5 vaccine in healthy individuals in the United States; 2. To perform a phase I dose-escalation study to assess the safety and immunogenicity of the rAd49 vaccine in healthy individuals in the United States; and 3. To perform a phase II study to assess the safety and immunogenicity of the heterologous rAd35k5-rAd49 prime-boost regimen in healthy individuals in the developing world.